The way in which photons travel through biological tissues and subsequently become scattered or absorbed is a key limitation for traditional optical medical imaging techniques using visible light. In contrast, near-infrared wavelengths, in particular those above 1000 nm, penetrate deeper in tissues and undergo less scattering and cause less photo-damage, which describes the so-called “second biological transparency window”. Unfortunately, current dyes and imaging probes have severely limited absorption profiles at such long wavelengths, and molecular engineering of novel NIR-II dyes can be a tedious and unpredictable process, which limits access to this optical window and impedes further developments. Two-photon (2P) absorption not only provides convenient access to this window by doubling the absorption wavelength of dyes, but also increases the possible resolution. This review aims to provide an update on the available 2P instrumentation and 2P luminescent materials available for optical imaging in the NIR-II window.